Characterization of the mevalonate kinase 5'-untranslated region provides evidence for coordinate regulation of cholesterol biosynthesis.
Using a probe derived from the 5'-untranslated region of the human mevalonate kinase (MK) cDNA, we screened a lambda gt 11 genomic library and obtained a single clone containing the 5' untranslated region of the gene. Nucleotide sequencing identified several putative regulatory elements, including two Sp1 (GC box) elements and a CCAAT box. A canonical TATA box was not detected. Directly adjacent to one Sp1 element was a sterol regulatory element (SRE), 5'-CACCCCAG-3', which was a 7/8 base pair match to the consensus sequences identified in the genes encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase and reductase, and the LDL receptor. There was no Sp1 element upstream of the SRE. Northern blot analysis in human CRL1508T cells revealed that quantities of MK poly A+ RNA increased for cells grown in the presence of lipid-deficient calf serum, and further increased upon addition of 1 microM lovastatin. Primer extension analysis with human poly A+ RNA suggested at least 4 transcription initiation sites downstream from the CCAAT box. To assess sterol responsiveness of transcription initiation, a 1.4 kb genomic fragment upstream of the translational start site was fused to the pSV2cat vector for transient expression in COS-7 cells, with chloramphenicol acetyltransferase (CAT) as the reporter gene. This construct demonstrated modest levels of CAT expression which was induced > 2-fold when cells were grown in lipoprotein-deficient calf serum. Our data provide further evidence for coordinate regulation of cholesterol biosynthesis in response to sterol.
Bishop
R W
RW
Geron Corporation, Menlo Park, California 94025, USA.
Chambliss
K L
KL
Hoffmann
G F
GF
Tanaka
R D
RD
Gibson
K M
KM
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D019556:COS Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002784:Cholesterol / Q000096:biosynthesis*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D008074:Lipoproteins / Q000172:deficiency / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000235:genetics
D008074:Lipoproteins; D012333:RNA, Messenger; D002784:Cholesterol; D008148:Lovastatin; D015500:Chloramphenicol O-Acetyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase